This is a proposal to explore the mechanisms of long-term tolerance of autoreactive CD4 T-cells induced by soluble MHC II/Fc/peptide chimeras. Our preliminary results indicated that short therapy with MHC II/Fc/peptide chimeras of prediabetic mice induced regulatory mechanisms responsible for tolerance to a self autoantigen, and offered protection against the disease onset for as long as 8 months. Using a double transgenic mouse model for insulin-dependent diabetes mellitus (IDDM) in which the autoreactive T-cells specific for a defined self epitope were not deleted by thymic selection, and they were not tolerized in periphery, we propose to explore the cellular, and molecular mechanisms induced by MHC II/Fc/peptide chimera leading to antigen-specific long-term tolerance. Manipulation of the antigen-specific tolerance to immunodominant autoreactive epitopes in the neonatal life, may soon lead to novel strategies aimed at arresting the epitope spreading in autoimmune diseases. Deciphering the cellular and molecular basis of these mechanisms may also offer a better understanding of why antigen-specific long-term tolerance can be easily induced in the neonatal life, but not in the adult life. In the first part of this application we will (1) investigate the ability of MHC II/Fc/peptide chimera to induce protective Th2 memory responses and regulatory/suppressor cells during particular age-windows, (2) characterize immunophenotypically and functionally the fate of these cells in adulthood, and (3) define their protective capacity against autoimmune diabetes in the IDDM double transgenic mouse model. In the second part, we will (1) explore the biochemical nature of several transductional and transcriptional events that can be differentially induced by MHC II/Fc/peptide chimera in early life, and that may affect the fate of Th2 and regulatory/suppressor cells in adulthood, and (2) investigate the biochemical nature of tolerogenic signals induced by regulatory/suppressor cells in the autoreactive T-cells in the target organ. Satisfactory results of this study will expand our area of investigations on the human MHC II/Fc/peptide-like reagents carrying immunodominant autoreactive epitopes relevant for human autoimmune diseases. The antigen-specific tolerogenicity of these reagents will be evaluated in in vitro systems, and in double transgenic mice deficient for the murine MHC class II, and expressing human HLA-DR* alleles.